Hereditary hemochromatosis is one of the most common inherited disorders and complications of cirrhosis are the most common cause of death in these patients. Hepatic fibrogenesis is mediated by the activation of lipocytes, a process which is characterized by a dramatic phenotypic transformation, wherein lipocytes acquire myofibroblastic features and an enhanced capacity to synthesize collagen. Little is known about the mechanism(s) which initiate the process of lipocyte activation. The long- term goals of this research project are to gain a better understanding of the initiating factors in lipocyte activation, with emphasis placed on the potential roles of oxidative stress, products of Kupffer cells, and intracellular signaling. In experimental iron overload, hepatocellular oxidative stress is accompanied by lipocyte activation and fibrosis adjacent to iron-loaded Kupffer cells. We now propose a series of experiments designed to test the key hypotheses linking oxidative stress, activation of Kupffer cells, and initiation of lipocyte activation. In Specific Aim 1, we will test the hypothesis that chronic iron overload activates the transcription factors, AP-1 and NF-kappaB, in Kupffer cells in vivo, and that these Kupffer cells produce mediators (TGF-beta, TNF- alpha) that can initiate activation of quiescent lipocytes. In Specific Aim 2, we will determine whether phagocytosis of damaged iron-loaded hepatocytes by Kupffer cells results in release of products (TGF-beta, TNF-alpha) which can initiate lipocyte activation. In Specific Aim 3, we will test the hypothesis that oxidative stress can directly induce the activity of AP-1 and NF-kappaB in Kupffer cells in vitro, and that oxidative stress increases the production of TGF-beta and TNF-alpha. In order to better understand the process of lipocyte activation, in Specific Aim 4 we will test the hypothesis that protein kinase C (PKC) plays a role in the initiation and perpetuation of lipocyte activation. These proposed experiments will dramatically expand our knowledge of the initiation of lipocyte activation, a key process in hepatic fibrogenesis. Apart from the obvious clinical relevance to chronic iron overload conditions in humans, these investigations have much broader significance in enhancing our understanding of the relationship between oxidative stress, activation of Kupffer cells and lipocyte activation in hepatic fibrogenesis.